It s in the Details Problems with Blood Bank Testing You Never Saw Coming

Transcription

1 It s in the Details Problems with Blood Bank Testing You Never Saw Coming Heart of America April 17, 2018 Presented by Rebecca Bullock, MT(ASCP)SBB Technical Director, Quotient Biodiagnostics

2 I have come to realize that the extras around the testing saline, tubes, temperature, washes, etc. which seem so fundamental, can really throw a kink in the works.

3 Objectives Review how simple, basic items used in Immunohematology testing, such as supplies and standard protocols, affect the results we obtain. Explain the historical, scientific origins of our current standard testing protocols in the blood bank. Discuss unusual cases where unexpected variables caused problems in blood bank testing, as well as the techniques and logic used in the resolution of the cases presented

4 Essential processes are all composed of tasks or activities, some of which actually contribute to the process, while others exist just because we ve always done it that way some of these activities add value to the process and some of them don t Walters, Lisa M. Introducing the Big Q: A Practical Quality Primer. AABB Press, Page 107

5 To manage a system of processes effectively, the facility must understand how its processes interact and what cause and effect relationships exist between them AABB Technical Manual, Nineteenth Edition. AABB Press, Pg. 4.

6 Drivers of Change Mistakes made in your own lab Investigations made in your lab Mistakes made in other facilities Citations from inspections, your own or others Published cases Published research

7 Antigen typing Manufacturer Quotient Anti - N Manufacturer Other Manufacturer Anti - N Instructions 1 drop reagent 1 drop of 2-4% red cell suspension; Mix well Incubate C Spin / read 1 drop reagent 2 drops of 2% red cell suspension; Mix well Incubate C Do Not Spin / read Positive Control M+N+ cell Negative Control M+N- cell Neg

8 Investigation Major difference in testing was length of incubation 5 minutes versus 30 minutes Technologists generally removed reagents from the refrigerator and used them immediately. Quotient Anti-N sera was not being tested at the correct incubation temperature. Package insert states False positive or false negative results can occur due to improper reaction temperature

9 Antigen typing Manufacturer Quotient Anti - N Manufacturer Other Manufacturer Anti - N Instructions 1 drop reagent 1 drop of 2-4% red cell suspension; Mix well Incubate C Spin / read 1 drop reagent 2 drops of 2% red cell suspension; Mix well Incubate C Do Not Spin / read Positive Control M+N+ cell Negative Control M+N- cell Neg Neg

10 Inspection Citation AABB inspection on the West Coast. Citation for performing testing using an antisera that was supposed to be tested at room temperature, defined in the package insert as o C. The laboratory was warm. Inspector asked about the room temperature. Thermometer read 24.5 o C

11 Room Temp Temperature Range

12 Room Temp Temperature Range Sweet spot = o C

13 Processes to Prove In Control Room temperature probe hooked up to electronic monitoring system (along with refrigerators and freezers) to monitor room temperature in the IRL. Thermometers at each desk, and recording the room temperature each time ran a room temperature test. ARC - checking the package insert directions before testing each and every time. Only use Phosphate buffered saline.

14 Temperatures aabb Technical Manual, Nineteenth Edition. Bethesda, MD: AABB, General Laboratory Methods Section Temperatures Whenever specific incubation or storage temperatures are given, the following ranges are considered satisfactory: Stated Temperature Acceptable Range 4 C 2-8 C Room Temperature C 37 C C 56 C C

15 Unusual Quotes from Package Inserts optical aids Do not use any optical aid to examine the test results. Do not examine microscopically. Optical Aid (opt) The use of an optical aid for agglutination reading must be validated by the user.

16 Unusual Quotes from Package Inserts optical aids Resuspend the cells completely and examine macroscopically without the use of magnifying devices, i.e., hand lens, agglutination viewer or microscope, for agglutination. Use of such devices may lead to misinterpretation of negative reactions as positive reactions.

17 Unusual Quotes from Anti-sera Inserts Do not centrifuge at any time. Centrifuge 60 seconds at x g. Adding 2 drops of reagent may enhance reactivity. Incubating for the upper end of the time range may enhance reactivity. Materials Required: 10x75 mm or 12x75 mm glass test tubes

18 Unusual Quotes from Anti-sera Inserts reactions of 1+ or weaker (test or control cells) should be investigated before a phenotype is assigned, as they may be an indication that the environmental temperature, centrifugation speed or time, or the volume of reagent or cell suspension used are not optimum or that the reagent is deteriorating. NOTE: Hemolysis, if obtained, should not be interpreted as a positive test. Hemolysis may indicate that the reagent has become contaminated with bacteria.

19 Unusual Quotes from Anti-sera Inserts Washed red blood cells may demonstrate better reactions Add 1 drop of an approximately 3-4% suspension of the red blood cells to be tested (previously washed at least 1 time and resuspended in saline) Anti-X must be used only with red blood cells suspended in isotonic saline. The optimal isotonic saline for most antibodies is ph Use of isotonic saline with a low ph may cause decreased sensitivity.

20 Isotonic Saline Isotonic applies to any solution that has the same osmotic pressure as blood. Isotonic saline (normal saline) is 0.9% solution of salt in H 2 O

21 Saline Isotonic Blood Bank Saline Isotonic NaCl solution % weight to volume ph range of Phosphate Buffered Blood Bank Saline Isotonic NaCl solution % weight to volume ph range of (may range as high as 7.5) Buffered with phosphate

22 Saline Braun Saline 0.9% weight to volume Sterile saline solution Used for irrigation of wounds and IV solutions ph range of (goal seemed to be 5.0 or 5.6) Available in small volumes of 250, 1000 or 3000 ml

23 Saline Bruce, M. et.al. A serious source of error in antiglobulin testing. Transfusion, 1986; 26: Abstract: The investigation of a failure of proficiency showed that certain saline solutions are inappropriate for use in blood group serology tests. In particular, it was found that solutions of unexpectedly low ph and/or those autoclaved and stored in plastic containers could severely compromise the sensitivity of the antiglobulin test when used as wash solutions.

24 Saline Bruce, M. et.al. A serious source of error in antiglobulin testing. Transfusion, 1986; 26: Evaluated 26 saline solutions from 10 blood banks from multiple sources Range of saline ph Some examples of Anti-D, Anti-S, Anti-s, Anti-Fy a, Anti-Jk a, Anti-Mi a and Anti-V w failed to react or reacted weakly, when AHG testing was performed using saline with ph of < 6.5

25 Abstract: The observed loss of sensitivity ranged from a reduction in titration score to a complete failure in the detection of clinically significant blood group antibodies

26 Saline Bruce, M. et.al. A serious source of error in antiglobulin testing. Transfusion, 1986; 26: Low ph decreased AHG reactivity by two separate mechanisms. Dissociation of bound IgG during washing Exposure of gamma globulin to acid causes these molecules to swell symmetrically as the ph is lowered, producing conformational changes which renders the IgG molecules incapable of reacting with anti-igg.

27 Why? Intact red cells maintain normal shape when the red cell has an internal ph of Red cells will invaginate at lower ph values. Suspension in saline of more extreme ph will affect the red cell membrane curvature ph < 6.0, becomes negative (stomatocytic) ph >8.0, curvature becomes positive (echinocytic) Effects of ph changes of stock normal saline solution on 5% red cell suspension. Martin, GL, et. al.

28 Why? Red cells have negative charge at ph = 7 - Antibody molecules have weakly positive charge at + ph of Enhances attraction between antigens and antibodies during first stage of agglutination / sensitization Acid elution - decrease ph, disassociate antibody

29 Phosphate-Buffered Saline Bruce, M. et.al. A serious source of error in antiglobulin testing. Transfusion, 1986; 26: Again, from the abstract, they concluded that improved standardization and sensitivity (in serological tests) could be achieved by using phosphate-buffered saline ph It is recommended that unbuffered saline solutions of ph less than 6.0 should not be used for serological testing.

30 Saline Rolih, S. et.al. Antibody detection errors due to acidic or unbuffered saline. Immunohematology, 1993 Vol 9, pg Study to replicate the findings from 1986, using unbuffered and buffered saline, and to determine if ph had an effect on solid phase testing. ph range tested was

31 Saline Rolih, S. et.al. Antibody detection errors due to acidic or unbuffered saline. Immunohematology, 1993 Vol 9, pg Conclusion: We also support the suggestion of these authors that saline solutions used in antibody detection tests be considered as important as the potentiating media, reagent red cell type, or antiglobulin reagent employed. Thus the ph of saline should be strictly controlled at when either solid phase testing or hemagglutination testing is performed.

32 Saline AABB Technical Manual, 19 th edition Page 486. Sources of False-Negative results in Antiglobulin Testing /Saline The low ph of saline solution can decrease the sensitivity of the test. 2 The optimal ph of saline wash solution for most antibodies is 7.0 to 7.2.

33 Unusual Quotes from Anti-sera Inserts Add 1 volume of red blood cells suspended to 2-4% in unbuffered isotonic saline. As this reagent reacts optimally at ph 8.5 and is extremely sensitive to ph, test red blood cells should be suspended in unbuffered medium. All red blood cells suspended in buffered medium e.g. Alsever s solution, should be washed at least once and resuspended in unbuffered saline prior to use. From Quotient Anti-M insert. Although many M antibodies may be enhanced by acidic ph, this antibody is monoclonal

34 Monoclonal antibodies Produced from a single immune cell line All antibody molecules are identical Highly specific and generally high titre No variation from batch to batch Large quantities can be made Majority are of murine (mouse) origin Some (anti-ds) are of human origin

35 Polyclonal antibodies Usually of human origin Same specificity but produced by different clones of antibody producing cells Each molecule is NOT identical Each batch needs to be standardized to ensure same reactivity Each batch needs to be tested to ensure no other contaminating antibodies are present

36 The Age of Monoclonal Anti-sera Monoclonal anti-sera are not like polyclonal sera Monoclonal sera have been grown and manufactured. They may have unusual incubations, unusual spin times, specific room temp requirements, specific saline requirements Monoclonal antibodies are all from the same clone and directed at the same epitope they have no tolerance. To see the specificity, you have to follow the directions, exactly.

37 Know Your Monoclonal Case Study Mom typed as RhD negative by Gel testing on the ProVue. Baby typed as RhD negative by Gel testing on the ProVue. Cord samples are also tested in tube, at AHG Sample MOM Neg ABD Gel Card D type Quotient Anti-D IS / AHG CORD Neg 0 / 4+

38 Know Your Monoclonal Case Study Mom typed as RhD negative by Gel testing on the Provue. When tested in the tube, was Weak D at AHG. Baby typed as RhD negative by Gel testing on the ProVue. Cord samples are also tested in tube, was Weak D at AHG Sample ABD Gel Card D type Quotient Anti-D IS / AHG MOM Neg 0 / 4+ CORD Neg 0 / 4+

39 Know Your Monoclonal Case Study FDA requirement for Tube reagents: Anti-D must NOT detect Partial RhD Category VI on immediate spin. Anti-D MUST detect Partial RhD Category VI at the antiglobulin phase of testing (will look like a Weak D). This is usually carried out with a combination of monoclonal antibodies: IgM clone for the immediate spin + IgG clone for AHG. Ortho Monoclonal A/B/D Gel Card states: Most weak D antigen expressions will be detected as weak positive reactions with this reagent. However the partial D VI epitope variant of the D antigen will not be detected with this monoclonal reagent. Anti-D is derived from a single cell line, MS-201, a monoclonal human IgM Anti-D.

40 Know Your Monoclonal Case Study Sample from Mom Tested using Advanced Partial RhD Typing Kit

41 Know Your Monoclonal Case Study Sample from Cord Blood Tested using Advanced Partial RhD Typing Kit

42 Plastic versus Glass Tubes Detail # 4 Widely accepted, although little is documented The use of plastic rather than glass is especially a concern when using monoclonal antibodies, where to a certain extent every antibody is identical. This means that the overall charge on the antibodies is within a very narrow range. If the antibody charge is incompatible with the charge on the plastic (the surface of which is more open and charged than glass), the antibody can be completely adsorbed. Glass is much more inert.

43 Plastic versus Glass Tubes Alba Bioscience experience One monoclonal antibody that requires a different filter because when processed with the company s filter of choice, the antibody is completely removed during filtration, due to binding to the membrane and housing of the filter. Also seen sharp decline of potency of some monoclonal antibodies when stored in plastic.

44 Washing / AHG / Check cells GREEN Anti-Human Globulin or GREEN Anti-IgG Adequate washing Weak or strong IgG Sensitized Cells

45 Validation of Laboratory Equipment Cell Washers Great expansion of validation process to include Adequate wash volume Adequate decant cycle; residual saline left on cell button Appropriate spin times for each type of test, (well formed button that is easily dispersed) Annual performance of cell button test Monthly measurement of wash volume Maintenance: changing pump tubing, or cleaning with bleach and rinsing multiple times

46 Cell Washer Maintenance Rygiel SA, Issitt CH, Fruitstone MJ. Destruction of the S antigen by Clorox (abstract). Transfusion 1983; 23: 410. Sodium Hypochlorite (Clorox) oxidizes the methionine residue at position 29 in the Ss sialoglycoprotein resulting in the loss of S antigen expression At levels of % Clorox in a 3% suspension, S+s+ RBCs become immediately non-reactive with anti-s; S+s- RBCs required % Clorox in PBS

47 Non-reactive / weak Coombs Control cells Issitt PD, Anstee DJ. Applied Blood Group Serology, Third Edition. Durham, NC: Montgomery Scientific Publications, Pg Washing of red cells prior to adding antiglobulin serum must be very thorough, as it actually constitutes a series of dilutions in which the unbound proteins remaining from the original serum or plasma are diluted in successive lots of wash saline. It is known that as little as 2mg of IgG/ml remaining at the end of the wash cycle can cause neutralization of antiglobulin serum when it is added.

48 Non-reactive / weak Coombs Control cells Issitt PD, Anstee DJ. Applied Blood Group Serology, Fourth Edition. Durham, NC: Montgomery Scientific Publications, Pg

49 Weak or Strong Check Cells

50 Git R Done 57 yr. old white female Diagnosis : Ovarian Cancer Anemia due to chemotherapy Patient has history of Anti-Fy a, Anti-E, Anti-V

51 Pre-transfusion Sample B Positive Antibody Screen Cell I 3+ Cell II 0 DAT Negative with Anti-IgG Antibody ID: Anti-Fy a only Anti-E, Anti-V not demonstrated at this time 2 units of E- Fy(a-) packed red blood cell units were crossmatched for outpatient transfusion on July 1.

52 July 1 10:43 1st unit issued and transfused 12:53 2nd unit issued and transfusion started 14:20 Patient had chills and nausea (after 300 ml) Pre Post Temp 97.5 Temp 97.7 BP 133/72 BP 191/86 Pulse 94 Pulse 101 Resp 18 Resp 18

53 Transfusion Reaction Workup Post Transfusion Reaction Sample Collected July 1 at 16:45 Plasma appearance = slight amber ABO/Rh = B Positive DAT = 1+ Anti-IgG = 0 Anti-C3b, -C3d Antibody ID in plasma = Anti-Fy a only

54 Appearance of the Plasma

55 Transfusion Reaction Workup Eluate: Anti-Fy a Crossmatches repeated with post-transfusion sample and pre-transfusion sample 1 st unit crossmatch = compatible 2 nd unit = incompatible (2+) at AHG using anti-igg Blood Bank Medical Director suggested clinical monitoring and follow-up. Patient admitted to hospital.

56 Transfusion Reaction Workup Repeat E and Fy a antigen types 1 st unit E- Fy(a-) 2 nd unit E- Fy(a+) (Fy a typing was 2+) Post Transfusion Urine collected day 2 (July 2) at 11:23 - Color Brown - Blood Large (performed on unspun urine) RBC/hpf

57 Other Lab Values Hgb Hct BUN Creat LDH Tbili DBili 6/ NT NT NT NT NT 7/1 16: /2 11: NT NT NT 7/3 04: NT NT NT Patient transfused 2 E- Fy(a-) V- packed RBCs (xmatch compatible) 7/4 11: Patient discharged to home

58 Root Cause Analysis 1 st unit was antigen typed and crossmatched by evening shift 2 nd unit was antigen typed and crossmatched by overnight shift Overnight shift tech is one of 5 techs covering the entire lab She used the cell washer to wash the tubes for the Fy a antigen type and also for the crossmatch After adding the Anti-IgG, she centrifuged the tubes and read the tubes after performing other tasks. Estimated time between adding Anti-IgG and reading tubes was a few minutes.

59 Root Cause Analysis Detail # 7 Question? Could the missed reactivity be due to the delay in reading the antiglobulin phase of testing?

60 Time elapsed between addition of Anti-IgG and reading reactions. Donor Cells Pos Control Fy(a+b+) Neg Control Fy(a-b+) 12FP12914 Fy(a+b-) 12FQ03355 Fy(a+b-) 12KE24101 Fy(a-b+) Immediate Spin 30 sec 60 sec 2 min 5 min w

61 Lessons Learned Root Cause Analysis is helpful to determine when, where, who, what and how an error was made and to make changes to prevent future errors The manufacturers instructions for use of all antisera must be read and followed carefully When performing IAT, don t delay reading tubes after adding Anti-IgG (AHG)

62 The greatest blunders, like the thickest ropes, are often compounded of a multitude of strands." Victor Hugo, French novelist and poet Mistakes, obviously, show us what needs improving. Without mistakes, how would we know what we had to work on? Peter McWilliams, Life 101

63 Rebecca Bullock, MT(ASCP)SBB Technical Director quotientbd.com